The capacity of cells to adapt to stresses such as heat shock, chemical insult or nutrient deprivation, to suppress misfolding and aggregations of proteins, and to degrade aberrant polypeptides is essential for viability. Such functions are effected by "molecular chaperones", proteins which have specific mechanisms for modulating synthesis, folding, renaturation and degradation of proteins in cells. The long term goal of this work is to understand the structures and biochemical mechanisms of molecular chaperones. The Clp/Hsp100 proteins are a diverse family of ATP-dependent molecular chaperones. They include the bacterial HslU which interfaces to the protease HslV to form the "prokaryotic proteasome", yeast Hsp104 which modulates formation and dissolution of fibrils of the prion-like [PSI+] factor, and mammalian torsins, in which mutations are associated with neurological dystonias. Using HslU as a prototype Clp/Hsp100 protein, the goal is to understand the molecular mechanisms by which substrates are selected, unfolded and translocated through a narrow channel into the proteolytic cavity of HslV. This will be accomplished through enzymatic assays of polypeptide degradation and ATP turnover, ensemble solution and single molecule fluorescence studies designed to measure the step size and step rate of the translocation process, and structural studies to correlate the polypeptide translocation activity with ATP-driven conformational changes in the HslUV complex. A clear understanding of the mechanism of protein folding/unfolding of one Clp/Hsp100 protein can provide insights into related proteins, the malfunction of which is correlated with protein aggregation-related pathologies. Little is known about the mechanism by which membrane proteins are folded and assembled. In gram negative bacteria, specific periplasmic chaperones facilitate correct maturation of outer membrane porins. Using recent information on the structure and peptide binding specificity of one of these chaperones, E. coli SurA, the goal is to apply structural (NMR and crystallography) and biophysical methods to map the peptide binding activity of the chaperone, to correlate the in vitro peptide binding activity with in vivo interactions with membrane proteins, and ultimately to develop an in vitro assay of bacterial porin folding and assembly that will allow direct biochemical assay of SurA and related proteins.